用基因组改组技术改良发酵木糖酵母Candida tropicalis XY-19的耐乙醇性能

Candida tropicalis XY-19是一株具有优良乙醇发酵性能的发酵木糖酵母,其发酵葡萄糖产乙醇性能与目前酒精工业生产菌种--安琪酒精酵母相近,但XY-19的耐乙醇性能远比安琪酒精酵母差,XY-19在含有超过7%（v/v）乙醇的培养基中不能生长。以XY-19为出发菌株,经紫外线诱变获得了5株能在7.5%（v/v）乙醇的培养基中旺盛生长的突变株,经Co-60诱变获得了8株能在含8%（v/v）乙醇的培养基中旺盛生长的突变株。然后,以紫外线诱变得到的5株菌和Co-60诱变得到的8株菌及耐乙醇性能较好的酿酒酵母（S.cerevisae Angel,S.cerevisae4608和S.cerevisae172）为出发菌株,经过4轮Genome shuffling结合木糖乙醇梯度平板的筛选,获得了4株（G3-13,G3-18,G3-57和G3-60）能够在12%乙醇平板上生长的菌株,其乙醇耐受性比野生菌株XY-19提高了71%,为将XY-19进一步开发成纤维质乙醇发酵的生产菌种奠定基础。本研究结果进一步体现了Genome shuffling技术在改良如乙醇耐受性等多基因控制性状上的突出优势,为工业生产菌种的快速有效改良提供了一种有效的方法。

Improvement of Ethanol Tolerance of Xylose-fermenting Yeast Candida tropicalis XY-19 by Using Genome Shuffling

The ethanol production capability from glucose of xylose-fermenting yeast Candida tropicalis XY-19 was similar to S.cerevisae Angel,a commercial ethanol producer popularly used in China.But the ethanol tolerance of XY-19 was much lower than S.cerevisae Angel.XY-19 could not grow in YPD broth containing ethanol over 7% （v/v）.XY-19 cells were exposed to UV irradiation and 5 mutant strains which could grow in media containing 7.5% （v/v） of ethanol were obtained.The UV irradiated mutants were mutagenized with Co-60 and 8 mutant strains which could grow in media containing 8% （v/v） of ethanol were obtained.The UV mutants,Co-60 mutants and 3 strains of S.cerevisae Angel,S.cerevisae 4608 and S.cerevisae 172 with higher ethanol tolerance were selected as the starter for genome shuffling.After four rounds of genome shuffling and selecting on ethanol gradient plates with xylose as sole carbon source,four shuffled strains （G3-13,G3-18,G3-57 and G3-60） which could grow in YPD broth containing 12% （v/v） of ethanol were obtained.The ethanol tolerances of the 4 shuffled strains were 71% higher than that of the wild-type strain of XY-19.The research results suggest that XY-19 has the potential to be able to develop into a commercial cellulosic ethanol producer.The research results also show the advantage of genome shuffling in improvement of multigene-controlled phenotypes such as ethanol tolerance.Genome shuf-fling is an effective method in rapidly improvement of strains used in industrial production.